Rat serum carboxylesterase. Cloning, expression, regulation, and evidence of secretion from liver.

Multiple forms of carboxylesterase have been identified in rat liver, and five carboxylesterases (designated hydrolases A, B, C, S, and egasyn) have been cloned. Hydrolases A, B, C, and egasyn all have a C-terminal consensus sequence (HXEL) for retaining proteins in the endoplasmic reticulum, and these carboxylesterases are found in rat liver microsomes. In contrast, hydrolase S lacks this C-terminal consensus sequence and is presumed to be secreted. In order to test this hypothesis, a polyclonal antibody was raised against recombinant hydrolase S from cDNA-directed expression in Escherichia coli. In addition to hydrolases A, B, and C (57-59 kDa), this antibody recognized a 67-kDa protein in rat liver microsomes and a 71-kDa protein in rat serum. The 71-kDa protein detected in rat serum was also detected in the extracellular medium from primary cultures of rat hepatocytes. Non-denaturing gel electrophoresis with staining for esterase activity showed that a serum carboxylesterase comigrated with the 71-kDa protein. Immunoprecipitation of the 71-kDa enzyme from rat serum decreased esterase activity toward 1-naphthylacetate and para-nitrophenylacetate. The 71-kDa protein immunoprecipitated from rat serum had an N-terminal amino acid sequence identical to that predicted from the cDNA encoding hydrolase S, providing further evidence that hydrolase S is synthesized in and secreted by the liver. The levels of the 67-kDa protein in rat liver microsomes and the levels of the 71-kDa protein in rat serum were co-regulated. Deglycosylation of microsomes and serum converted the 67- and 71-kDa proteins to a 58-kDa peptide, which matches the molecular mass calculated from the cDNA for hydrolase S. These results suggest that the 67-kDa protein in liver microsomes is a precursor form of hydrolase S that undergoes further glycosylation before being secreted into serum. In rats, liver appears to be the only source of hydrolase S because no mRNA encoding hydrolase S could be detected in several extrahepatic tissues. Serum carboxylesterases have been found to play an important role in lipid metabolism and detoxication of organophosphates, therefore, the secretion of hydrolase S and the modulation of its expression by xenobiotics may have physiological as well as toxicological significance.